Comprehensive Structural Analysis of the Genome of Red Clover (Trifolium pratense L.)

Abstract

With the aim of establishing the basic knowledge and resources needed for applied genetics, we investigated the genome structure of red clover Trifolium pratense L. by a combination of cytological, genomic and genetic approaches. The deduced genome size was approximately 440 Mb, as estimated by measuring the nuclear DNA content by flow cytometry. Seven chromosomes could be distinguished by microscopic observation of DAPI stained prometaphase chromosomes and fluorescence in situ hybridization using 28S and 5S rDNA probes and bacterial artificial chromosome probes containing microsatellite markers with known positions on a genetic linkage map. The average GC content of the genomes of chloroplast, mitochondrion and nucleus were shown to be 33.8, 42.9 and 34.2%, respectively, by the analysis of 1.4 Mb of random genomic sequences. A total of 26,356 expressed sequence tags (ESTs) that were grouped into 9339 non-redundant sequences were collected, and 78% of the ESTs showed sequence similarity to registered genes, mainly of Arabidopsis thaliana and rice. To facilitate basic and applied genetics in red clover, we generated a high-density genetic linkage map with gene-associated microsatellite markers. A total of 7159 primer pairs were designed to amplify simple sequence repeats (SSRs) identified in four different types of libraries. Based on sequence similarity, 82% of the SSRs were likely to be associated with genes. Polymorphism was examined using two parent plants, HR and R130, and 10 F(1) progeny by agarose gel electrophoresis, followed by genotyping for the primer pairs showing polymorphisms using 188 F(1) plants from the mapping population. The selected 1305 microsatellite markers as well as the previously developed 167 restriction fragment length polymorphism markers were subjected to linkage analysis. A total of 1434 loci detected by 1399 markers were successfully mapped onto seven linkage groups totaling 868.7 cM in length; 405 loci (28%) were bi-parental, 611 (43%) were specific to HR and 418 (29%) were specific to R130. Each genetic linkage group was linked to a corresponding chromosome by FISH analysis using seven microsatellite markers specific to each of the linkage groups as probes. Transferability of the developed microsatellite markers to other germplasms was confirmed by testing 268 selected markers on 88 red clover germplasms. Macrosynteny at the segmental level was observed between the genomes of red clover and two model legumes, Lotus japonicus and Medicago truncatula, strongly suggesting that the genome information for the model legumes is transferable to red clover for genetic investigations and experimental breeding.

Modern methods for genetic improvement of Trifolium pratense.

“…Arumuganathan and Earle (1991) performed flow cytometric measurement and revealed 2C genome size of 0.97 pg. Rizza et al (2007) Sato et al (2005) deduced a haploid genome size of 440 Mb, as estimated by measuring the nuclear DNA content using flow cytometry (two red clover plants with 0.89 and 0.91 pg/2C), with 1 pg of DNA equivalent to 980 Mb (Bennett et al 2000). Ellison et al (2006) and Vižintin et al (2006) reported results of detailed and comprehensive studies of the molecular phylogenetic framework for the genus Trifolium.…”

“…Phylogenetic relationship is a key factor for comparative genomics within the genus or family and for exchanging knowledge in relation to the various species (Sato et al 2010). Sato et al (2005) analysed a karyotype of the red clover genome by microscopic observation of prometaphase chromosomes. The lengths of the prometaphase chromosomes ranged from 5.1 to 7.4 μm, and uneven condensation patterns were observed that could be useful in chromosome identification.…”

“…The lengths of the prometaphase chromosomes ranged from 5.1 to 7.4 μm, and uneven condensation patterns were observed that could be useful in chromosome identification. The genome structure of red clover using fluorescence in situ hybridization (FISH) was investigated by Sato et al (2005) and Kataoka et al (2012). Some chromosomes were distinguished using 26S and 5S rDNA probes.…”

“…R130 originated from a Russian variety and a wild variety from the Archangelsk region which exhibits late flowering, semi-prostrate shape, white flower colour, and small leaves. Sato et al (2005) developed a large collection of simple sequence repeat (SSR) markers from four types of genomic and cDNA libraries constructed from the above-mentioned genotypes HR and R130. A total of 1434 loci were detected by SSRs from this collection and another 167 RFLP loci were mapped onto seven linkage groups.…”

“…A mappingbased approach was used to identify SSRs closely linked to the GSI-locus (Riday & Krohn 2010). SSR markers developed by Sato et al (2005) were used and a bi-parental cross was initiated in which the parents were known to have one self-incompatibility allele (S-allele) in common. S-allele genotypes of 100 progenies were determined through test crosses and pollen compatibility.…”